Previous research supported by this grant led to the discovery of phosphoinositide 3-kinase (PI 3-kinase), and the realization that five different phosphoinositides exist in mammalian cells (PtdIns-4-P, PtdIns-4- P, Ptdins-3,4-P2, Ptdins-4,5-P2 and Ptdins-3,4,5-P3) rather than two as previously thought. During the past granting period, cDNA clones of several of the enzymes that make these lipids have been cloned and these enzymes have been found to play unexpected critical roles in a host of cellular functions. The hypothesis guiding this proposal is that subdomains of the Ptdins kinases unique to specific isoforms allow independent control of production of these phosphoinositides at distinct compartments of the cell for distinct purposes. The goal of this proposal is to compare the structures of the different PtdIns kinases and dissect the domain structures that give them their unique properties. The specific aims are to 1) characterize a PtdIns 4-kinase we recently cloned, 2) obtain a cDNA clone of a PtdIns-4-P 5-kinase that associates with rac, 3) determine the structural basis for association of PI 3- kinase, PtdIns 4-kinase and PtdIns-4-P 5-kinase with cellular regulators, 4) determine the structural basis for lipid kinase activity and for an unexpected protein kinase activity intrinsic to PI 3-kinase and 5) determine the enzymatic activities intrinsic to a family of PI 3-kinase homologues that includes the recently- discovered Ataxia-Telangiectasia gene.